A significant fraction of newly synthesized proteins translocate into the endoplasmic reticulum (ER). Once associated with this compartment, nascent polypeptides are post- translationally processed, acquire their native confirmations, and are sorted for delivery to other organelles or to the extracellular milieu. However, disease-causing mutations may compromise protein folding and maturation, which in turn generate aggregation-prone species. To off-set the catastrophic effects that accompany the accumulation of defective polypeptides, these substrates can be selected, delivered back to the cytoplasm, and then degraded via a process defined in the Brodsky laboratory and termed ER associated degradation, or ERAD. The long- term goal of research in the laboratory is to understand how disease-associated ERAD substrates are identified and destroyed so that specific factors or steps in this pathway can be modulated to prevent disease onset. As a first step toward this goal, yeast expression systems for ERAD substrates linked to specific maladies have been generated. Model substrates have also been constructed to explore how different classes of aberrant proteins are selected and routed for degradation. To complement these approaches, an in vitro assay that recapitulates the ubiquitination of membrane-embedded substrates was developed. Using these tools, the proposed studies will first examine whether there is a direct link between the aggregation propensity of a membrane protein and its selection for ERAD. Next, the membrane-associated components that maintain substrate solubility during ERAD will be identified and characterized. Secreted proteins that traffic beyond the ER-and even some ERAD escapees-can be captured and destroyed in the vacuole/lysosome. The factors involved in deciding whether a protein in the late secretory pathway should be sorted to its final destinations or should be delivered to the vacuole/lysosome are poorly defined. To better characterize this pathway, a genetic platform was developed. Recent data demonstrate that members of the a-arrestin family play a key role during post-ER degradation. A molecular dissection of how these proteins function and with which partners they operate will be undertaken using biochemical methods. A new cell surface fluorescence assay will then be used to generate complementary, quantitative data on plasma membrane residence when a-arrestin activity is altered. The outcome of these efforts will test new hypotheses, lead to deeper mechanistic insights into the etiology of protein conformational diseases, and characterize factors that may serve as novel therapeutic targets.